Synthesis of the Novel PARP-1 Inhibitor AG-690/11026014 and Its
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Acta Pharmacologica Sinica (2017) 38: 638–650 © 2017 CPS and SIMM All rights reserved 1671-4083/17 www.nature.com/aps Original Article Synthesis of the novel PARP-1 inhibitor AG- 690/11026014 and its protective effects on angiotensin II-induced mouse cardiac remodeling Guo-shuai FENG1, Cui-ge ZHU2, Zhuo-ming LI1, Pan-xia WANG1, Yi HUANG1, Min LIU3, Ping HE1, Lan-lan LOU2, Shao-rui CHEN1, *, Pei-qing LIU1, * 1Department of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, China; 2Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, China; 3Department of Pharmacology, Shandong University School of Medicine, Ji-nan 250012, China Abstract We previously identified AG-690/11026014 (6014) as a novel poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor that effectively prevented angiotensin II (Ang II)-induced cardiomyocyte hypertrophy. In the present study, we reported a new synthesis route for 6014, and investigated its protective effects on Ang II-induced cardiac remodeling and cardiac dysfunction and the underlying mechanisms in mice. We designed a new synthesis route to obtain a sufficient quantity of 6014 for this in vivo study. C57BL/6J mice were infused with Ang II and treated with 6014 (10, 30, 90 mg·kg-1·d-1, ig) for 4 weeks. Then two-dimensional echocardiography was performed to assess the cardiac function and structure. Histological changes of the hearts were examined with HE staining and Masson’s trichrome staining. The protein expression was evaluated by Western blot, immunohistochemistry and immunofluorescence assays. The activities of sirtuin-1 (SIRT-1) and the content of NAD+ were detected with the corresponding test kits. Treatment with 6014 dose- dependently improved cardiac function, including LVEF, CO and SV and reversed the changes of cardiac structure in Ang II-infused mice: it significantly ameliorated Ang II-induced cardiac hypertrophy evidenced by attenuating the enlargement of cardiomyocytes, decreased HW/BW and LVW/BW, and decreased expression of hypertrophic markers ANF, BNP and β-MHC; it also prevented Ang II-induced cardiac fibrosis, as implied by the decrease in excess accumulation of extracellular matrix (ECM) components collagen I, collagen III and FN. Further studies revealed that treatment with 6014 did not affect the expression levels of PARP-1, but dose-dependently inhibited the activity of PARP-1 and subsequently restored the activity of SIRT-1 in heart tissues due to the decreased consumption of NAD+ and attenuated Poly-ADP-ribosylation (PARylation) of SIRT-1. In conclusion, the novel PARP-1 inhibitor 6014 effectively protects mice against AngII-induced cardiac remodeling and improves cardiac function. Thus, 6014 might be a potential therapeutic agent for heart diseases.. Keywords: cardiac remodeling; PARP-1 inhibitor; 6014; PARylation; NAD+; SIRT-1 Acta Pharmacologica Sinica (2017) 38: 638–650; doi: 10.1038/aps.2016.159; published online 27 Feb 2017 Introduction an important component of the RAAS, could induce cardiac [2–6] Cardiac remodeling, manifested clinically as hypertrophy remodeling following sustained hypertension . (enlargement), fibrosis, and dysfunction of the heart resulting PARylation, induced by poly(ADP-ribose) polymerases from cardiac load or injury, is generally accepted as a deter- (PARPs), is a type of reversible posttranslational modifica - minant of the pathogenesis of heart failure (HF)[1]. The renin- tion impacting numerous cellular processes, eg, DNA repair, [7] angiotensin-aldosterone system (RAAS) plays a pivotal role in transcription, metabolism, or immune functions . PARP-1, the physiological and pathological processes of cardiac remod- the predominant member of the PARPs, accounts for approxi- [8] eling. It is also widely accepted that angiotensin II (Ang II), mately 90% of the cellular PARylation events . Once acti- vated, PARP-1 catalyzes protein PARylation, which results in the formation of polymerized ADP-ribose (PAR) dependently *To whom correspondence should be addressed. on NAD+ donor molecules, and subsequently the covalent E-mail [email protected] (Shao-rui CHEN); [9] [email protected] (Pei-qing LIU) attachment of PAR polymers to target proteins . Received 2016-09-26 Accepted 2016-11-24 PARP-1 is involved in pathological cardiac remodeling www.chinaphar.com Feng GS et al 639 and heart failure. Inhibition of PARP-1 prevents pathologi- eluted with a gradient of 5% to 95% solvent A for 25 min and cal cardiac remodeling induced by partial abdominal aortic detected at 254 nm; solvent A was CH3CN in double-deionized [10] 1 13 constriction . PARP-1-deficient mice are protected from Ang H2O. H NMR and C NMR spectra (AvanceIII, Burker) were II-induced cardiac hypertrophy[11]. Moreover, PARP-1 activa- recorded using TMS as an internal standard with a Burker - tion facilitates the pathogenesis of cardiac fibrosis[12] and the BioSpinUltrashield 400 NMR system. High-resolution mass development of heart failure[13, 14]. These findings indicate that spectra (HRMS) (LCMS-IT-TOF, Shimadzu) were recorded on PARP-1 could serve as a promising target for cardiac diseases, a Shimadzu LCMS-IT-TOF Instrument. and the pharmacologic inhibition of PARP-1 may be a poten- tial therapeutic strategy[15–17]. General procedure for the synthesis of 6014 A previous study from our laboratory identified AG-690/ (1) 4-chlorophenol (77.8 mmol), 2-(chloromethyl)oxirane (306.9 11026014(6014) as a novel PARP-1 inhibitor from the Specs mmol) and K2CO3 (231.5 mmol) were refluxed in acetone database (www.specs.net), which contains approximately (100 mL) overnight, cooled to room temperature and filtered. 200,000 compounds based on virtual screening. We also The filtrate was concentrated and extracted with toluene and reported that 6014 could significantly attenuate Ang II- water, and then the 2-((4-chlorophenoxy)methyl)oxirane (M1) induced cardiomyocyte hypertrophy[18]. To further deter - in toluene was purified by column chromatography. mine the in vivo cardiac protective effect of 6014, the present (2) 3-methyl-1H-purine-2,6(3H,7H)-dione (30 mmol) and study used C57BL/6J mice infused with Ang II as the model, AcONa (60 mmol) were dissolved in AcOH, and Br 2 was and elucidated the effects and underlying mechanisms of added dropwise and reacted for 2 h at 65 °C. 8-Bromo-3- this novel PARP-1 inhibitor. This study aimed to determine methyl-1H-purine-2,6(3H,7H)-dione (M2) was generated by whether 6014, as a PARP-1 inhibitor, has therapeutic potential cooling, filtering, water washing and drying in a vacuum. for cardiac remodeling. (3) M1 (31.0 mmol) and M2 (25.8 mmol) in ethanol (120 mL) were combined with trimethylamine (2.6 mmol) and refluxed Materials and methods for 3 h. 8-Bromo-7-(3-(4-chlorophenoxy)-2-hydroxypropyl)- Reagents 3-methyl-1H-purine-2,6(3H,7H)-dione (M3) was produced by The compounds 3-aminobenzamide (3-AB), 4’,6-diamidino- cooling, filtering, ethanol washing and drying in a vacuum. 2-phenylindole (DAPI) and Ang II were purchased from (4) M3 (8.15 mmol) and 2-mercaptoacetic acid (16.3 mmol) in Sigma-Aldrich (St Louis, MO, USA). Anti-PARP-1, anti- DMF (35 mL) were combined with NaHCO3 (32.6 mmol) and collagen I and anti-collagen III antibodies were provided reacted for 2 h at 85 °C. The reaction was stopped with water by Proteintech Group (Chicago, IL, USA). Anti-ANF and and then washed by methyl tertiary butyl ether. The products anti-FN antibodies were purchased from Santa Cruz Bio - in the water were combined with HCl (2.0 mmol) until the pH technology (Santa Cruz, CA, USA). Anti-BNP antibody was reached 3, and 2-((7-(3-(4-chlorophenoxy)-2-hydroxypropyl)-3- obtained from Merck Millipore (Bedford, Ohio, USA). Anti- methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)thio)acetic PAR antibody was obtained by Trevigen (Gaithersburg, MD, acid (M4) was yielded after filtering, water washing and dry- USA). Anti-SIRT-1, anti-lamin B1, anti-GAPDH antibodies ing in a vacuum. and all secondary antibodies for Western blot experiments (5) M4 (7.26 mmol) and ammonium chloride (21.7 mmol) were purchased from Cell Signaling Technology (Boston, in DMF (40 mL) was added to HATU (8.71 mmol) and DIEA MA, USA). An hematoxylin and eosin (HE) staining kit was (21.8 mmol) on ice and reacted overnight at room tempera- purchased from Santa Cruz Biotechnology (Santa Cruz, CA, ture. The products were precipitated by adding water and USA). A Masson’s staining kit and immunohistochemistry then filtering. 2-((7-(3-(4-chlorophenoxy)-2-hydroxypropyl)- reagents including Cy3-conjugated goat anti-mouse IgG (H+L) 3-methyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)thio) were purchased from Wuhan Goodbio Technology Co, Ltd acetamide (6014) was purified by preparative chromatography (Wuhan, China). RIPA lysis buffer and IP lysis buffer were (Varian, Agilent). purchased from Beyotime Biotechnology (Jiangsu, China). The The synthesized intermediates M1-M4 were structurally protease and phosphatase inhibitor mini tablets, BCA protein identified by 1H NMR or MS, and 6014 was structurally identi- assay kit, Bradford protein assay kit, and protein G-agarose fied by 1H NMR, 13C NMR and HRMS. beads were obtained from Pierce Biotechnology (Rockford, AL, USA). A nuclear extract kit was purchased from ACTIVE 2-((4-chlorophenoxy)methyl)oxirane (M1) 1 MOTIF (Carlsbad, NM, USA). The quick start Bradford dye Crystal oil, yield 62.0%; H NMR (400 MHz, CDCl3) δ 7.28–7.22 reagent was obtained from Bio-Rad (Richmond, VA, USA). A (m, 2H), 6.90–6.83 (m, 2H), 4.23 (dd, J=11.0, 3.0 Hz, 1H), 3.93 SIRT-1 deacetylase fluorometric assay kit was purchased from (dd, J=11.0, 5.7 Hz, 1H), 3.39–3.32 (m, 1H), 2.92 (t, J=4.5 Hz, CycLex Ltd (Nagano, Japan).